Nerve Growth Factor (NGF) is required for the survival and development of sensory and sympathetic neurons both in vivo and in culture. The molecular mechanisms of NGF action are presently unknown. It is the objective of this research proposal to develop new tools for the study of high-affinity NGF receptors which would help us understand the underlying mechanisms of NGF action. Discrete, quantifiable, electron-microscopic (EM) markers for occupied NGF receptors are not presently available. One major goal of this proposal is to synthesize biologically active derivatives of NGF that would permit quantiative EM investigations of the surface distribution and mobility of NGF receptors. I intend to synthesize ferritin conjugates of NGF using techniques recently developed for the study of insulin receptors. In addition, I will prepare biotinylated derivatives of NGF, which, with the appropriate secondary visualizing agents, would permit rapid and direct EM visualization of NGF receptors. Studies with biotinyl-NGF conjugates would also provide important new information on the structure-function relationships in NGF action. Furthermore, biotinyl-NGF could be used to bring about rapid, affinity purification of solubilized NGF receptors. Biochemical and immunofluorescent procedures will be used to synthesize and screen various NGF conjugates for biological activity and ability to bind to specific cell-surface receptors on cultured rat sympathetic neurons and on pheochromocytoma-derived, rat PC12 cells. In order to obtain information on human NGF receptors, I plan to study the characteristics of the NGF receptor on human melanoma cells. These studies are directly relevant to an understanding of human familial dysautonomia, where a defect either in NGF and NGF receptor may be involved. Finally, I intend to prepare monoclonal antibodies to NGF receptor in order to obtain additional, highly specific molecular probes of receptor structure and function.